Novel compounds

ABSTRACT

ANT5 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing ANT5 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

FIELD OF THE INVENTION

[0001] This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides, to their use in therapy andin identifying compounds which may be agonists, antagonists and/orinhibitors which are potentially useful in therapy, and to production ofsuch polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

[0002] The drug discovery process is currently undergoing a fundamentalrevolution as it embraces ‘functional genomics’, that is, highthroughput genome- or gene-based biology. This approach as a means toidentify genes and gene products as therapeutic targets is rapidlysuperceding earlier approaches based on ‘positional cloning’. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

[0003] Functional genomics relies heavily on high-throughput DNAsequencing technologies and the various tools of bioinformatics toidentify gene sequences of potential interest from the many molecularbiology databases now available. There is a continuing need to identifyand characterise further genes and their related polypeptides/proteins,as targets for drug discovery.

[0004] The ADP/ATP translocator, or adenine nucleotide translocator(ANT), is the most abundant mitochondrial protein. In its functionalstate, ANT is a homodimer of 30-kD subunits embedded asymmetrically inthe inner mitochondrial membrane. The dimer forms a gated pore throughwhich ATP is moved from the matrix into the cytoplasm. Three distincthuman ANT cDNAs, ANT1, ANT2, and ANT3, have been cloned to date.

SUMMARY OF THE INVENTION

[0005] The present invention relates to ANT5, in particular ANT5polypeptides and ANT5 polynucleotides, recombinant materials and methodsfor their production. In another aspect, the invention relates tomethods for using such polypeptides and polynucleotides, including thetreatment of congestive heart failure, ischaemic heart disease, cardiacarrhytmias, diastolic or systolic dysfunction, hypertrophiccardiomyopathy, stroke, hereinafter referred to as “the Diseases”,amongst others. In a further aspect, the invention relates to methodsfor identifying agonists and antagonists/inhibitors using the materialsprovided by the invention, and treating conditions associated with ANT5imbalance with the identified compounds. In a still further aspect, theinvention relates to diagnostic assays for detecting diseases associatedwith inappropriate ANT5 activity or levels.

DESCRIPTION OF THE INVENTION

[0006] In a first aspect, the present invention relates to ANT5polypeptides. Such peptides include isolated polypetides comprising anamino acid sequence which has at least 70% identity, preferably at least80% identity, more preferably at least 90% identity, yet more preferablyat least 95% identity, most preferably at least 97-99% identity, to thatof SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptidesinclude those comprising the amino acid of SEQ ID NO:2.

[0007] Further peptides of the present invention include isolatedpolypeptides in which the amino acid sequence has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to the amino acid sequence of SEQ ID NO:2 over theentire length of SEQ ID NO:2. Such polypeptides include the polypeptideof SEQ ID NO:2.

[0008] Further peptides of the present invention include isolatedpolypeptides encoded by a polynucleotide comprising the sequencecontained in SEQ ID NO:1.

[0009] Polypeptides of the present invention are believed to be membersof the calcium-sensitive adenine nucleotide translocator family ofpolypeptides. They are therefore of interest because when thecontractile activity of the heart is stopped by cardiac arrest orventricular fibrillation, about 60-70% of the oxygen uptake ceases,showing that most of the high-energy phosphate production by oxidativephosphorylation is directed toward contractile activity. Because the ANTdetermines the rate of ADP/ATP flux between the mitochondrion and thecytosol, it is a logical candidate for regulator of cellular dependenceon oxidative energy metabolism. Muscular contraction and relexation aredirectly regulated by both energy and calcium fluxes in the cytoplasm.Importantly, we have cloned a novel calcium-sensitive adenine nucleotidetranslocator. This novel calcium-sensitive adenine nucleotidetranslocator may be instrumental in the regulation of energy fluxes bycalcium, thus of muscle contractile function. These properties arehereinafter referred to as “ANT5 activity” or “ANT5 polypeptideactivity” or “biological activity of ANT5”. Also included amongst theseactivities are antigenic and immunogenic activities of said ANT5polypeptides, in particular the antigenic and immunogenic activities ofthe polypeptide of SEQ ID NO:2. Preferably, a polypeptide of the presentinvention exhibits at least one biological activity of ANT5.

[0010] The polypeptides of the present invention may be in the form ofthe “mature” protein or may be a part of a larger protein such as aprecursor or fusion protein. It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

[0011] The present invention also includes variants of theaforementioned polypeptides, that is polypeptides that vary from thereferents by conservative amino acid substitutions, whereby a residue issubstituted by another with like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tyr. Particularlypreferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacids are substituted, deleted, or added in any combination.

[0012] Polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0013] In a further aspect, the present invention relates to ANT5polynucleotides. Such polynucleotides include isolated polynucleotidescomprising a nucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, to theamino acid sequence of SEQ ID NO:2, over the entire length of SEQ IDNO:2. In this regard, polypeptides which have at least 97% identity arehighly preferred, whilst those with at least 98-99% identity are morehighly preferred, and those with at least 99% identity are most highlypreferred. Such polynucleotides include a polynucleotide comprising thenucleotide sequence contained in SEQ ID NO:1 encoding the polypeptide ofSEQ ID NO:2.

[0014] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence that has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to a nucleotidesequence encoding a polypeptide of SEQ ID NO:2, over the entire codingregion. In this regard, polynucleotides which have at least 97% identityare highly preferred, whilst those with at least 98-99% identity aremore highly preferred, and those with at least 99% identity are mosthighly preferred.

[0015] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to SEQ ID NO:1 overthe entire length of SEQ ID NO:1. In this regard, polynucleotides whichhave at least 97% identity are highly preferred, whilst those with atleast 98-99% identity are more highly preferred, and those with at least99% identity are most highly preferred. Such polynucleotides include apolynucleotide comprising the polynucleotide of SEQ ID NO:1 as well asthe polynucleotide of SEQ ID NO:1.

[0016] The invention also provides polynucleotides which arecomplementary to all the above described polynucleotides.

[0017] The nucleotide sequence of SEQ ID NO:1 shows homology with HumanBAC clone GS025M02 from 7q21-q22, complete sequence. Waterston, R.Direct Submission to GenBank. Submitted (12-SEP-1997) Department ofGenetics, Washington University, 4444 Forest Park Avenue, St. Louis, Mo.63108, USA. The nucleotide sequence of SEQ ID NO:1 is a cDNA sequenceand comprises a polypeptide encoding sequence (nucleotide 71 to 2095)encoding a polypeptide of 674 amino acids, the polypeptide of SEQ IDNO:2. The nucleotide sequence encoding the polypeptide of SEQ ID NO:2may be identical to the polypeptide encoding sequence contained in SEQID NO:1 or it may be a sequence other than the one contained in SEQ IDNO:1, which, as a result of the redundancy (degeneracy) of the geneticcode, also encodes the polypeptide of SEQ ID NO:2. Preferredpolypeptides and polynucleotides of the present invention are expectedto have, inter alia, similar biological functions/properties to theirhomologous polypeptides and polynucleotides. Furthermore, preferredpolypeptides and polynucleotides of the present invention have at leastone ANT5 activity.

[0018] The present invention also relates to partial or otherpolynucleotide and polypeptide sequences which were first identifiedprior to the determination of the corresponding full length sequences ofSEQ ID NO:1 and SEQ ID NO:2.

[0019] Accordingly, in a further aspect, the present invention providesfor an isolated polynucleotide which:

[0020] (a) comprises a nucleotide sequence which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% identity to SEQ ID NO:3 over the entirelength of SEQ ID NO:3;

[0021] (b) has a nucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% identity, to SEQ ID NO:3 over the entire length of SEQ ID NO:3;

[0022] (c) the polynucleotide of SEQ ID NO:3; or

[0023] (d) a nucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% identity, to the amino acid sequence of SEQID NO:4, over the entire length of SEQ ID NO:4;

[0024] as well as the polynucleotide of SEQ ID NO:3

[0025] The present invention further provides for a polypeptide which:

[0026] (a) comprises an amino acid sequence which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, most preferably atleast 97-99% identity, to that of SEQ ID NO:4 over the entire length ofSEQ ID NO:4;

[0027] (b) has an amino acid sequence which is at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to the amino acid sequence of SEQ ID NO:4 over theentire length of SEQ ID NO:4;

[0028] (c) comprises the amino acid of SEQ ID NO:4; and

[0029] (d) is the polypeptide of SEQ ID NO:4;

[0030] as well as polypeptides encoded by a polynucleotide comprisingthe sequence contained in SEQ ID NO:3.

[0031] The nucleotide sequence of SEQ ID NO:3 and the peptide sequenceencoded thereby are derived from EST (Expressed Sequence Tag) sequences.It is recognised by those skilled in the art that there will inevitablybe some nucleotide sequence reading errors in EST sequences (see Adams,M. D. et al, Nature 377 (supp) 3, 1995). Accordingly, the nucleotidesequence of SEQ ID NO:3 and the peptide sequence encoded therefrom aretherefore subject to the same inherent limitations in sequence accuracy.Furthermore, the peptide sequence encoded by SEQ ID NO:3 comprises aregion of identity or close homology and/or close structural similarity(for example a conservative amino acid difference) with the closesthomologous or structurally similar protein.

[0032] Polynucleotides of the present invention may be obtained, usingstandard cloning and screening techniques, from a cDNA library derivedfrom mRNA in cells of human heart, brain, uterus, mammary gland, lung,prostate, kidney, trachea, stomach, liver, placenta, testis, smallintestine, spinal cord, ovary, spleen, pancreas, thymus, aorta,leukocyte, skeletal muscle, adrenal, adipose, lymph node, colon,thyroid, bone marrow, bladder, salivary gland, appendix, using theexpressed sequence tag (EST) analysis (Adams, M. D., et al. Science(1991) 252:1651-1656; Adams, M. D. et al., Nature, (1992) 355:632-634;Adams, M. D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides ofthe invention can also be obtained from natural sources such as genomicDNA libraries or can be synthesized using well known and commerciallyavailable techniques.

[0033] When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself, or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions. For example, amarker sequence which facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

[0034] Further embodiments of the present invention includepolynucleotides encoding polypeptide variants which comprise the aminoacid sequence of SEQ ID NO:2 and in which several, for instance from 5to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted,deleted or added, in any combination.

[0035] Polynucleotides which are identical or sufficiently identical toa nucleotide sequence contained in SEQ ID NO:1, may be used ashybridization probes for cDNA and genomic DNA or as primers for anucleic acid amplification (PCR) reaction, to isolate full-length cDNAsand genomic clones encoding polypeptides of the present invention and toisolate cDNA and genomic clones of other genes (including genes encodingparalogs from human sources and orthologs and paralogs from speciesother than human) that have a high sequence similarity to SEQ ID NO:1.Typically these nucleotide sequences are 70% identical, preferably 80%identical, more preferably 90% identical, most preferably 95% identicalto that of the referent. The probes or primers will generally compriseat least 15 nucleotides, preferably, at least 30 nucleotides and mayhave at least 50 nucleotides. Particularly preferred probes will havebetween 30 and 50 nucleotides. Particularly preferred primers will havebetween 20 and 25 nucleotides.

[0036] A polynucleotide encoding a polypeptide of the present invention,including homologs from species other than human, may be obtained by aprocess which comprises the steps of screening an appropriate libraryunder stringent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof; and isolating full-lengthcDNA and genomic clones containing said polynucleotide sequence. Suchhybridization techniques are well known to the skilled artisan.Preferred stringent hybridization conditions include overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured, sheared salmon sperm DNA; followed by washing the filters in0.1×SSC at about 65° C. Thus the present invention also includespolynucleotides obtainable by screening an appropriate library understingent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof.

[0037] The skilled artisan will appreciate that, in many cases, anisolated cDNA sequence will be incomplete, in that the region coding forthe polypeptide is short at the 5′ end of the cDNA. This is aconsequence of reverse transcriptase, an enzyme with inherently low‘processivity’ (a measure of the ability of the enzyme to remainattached to the template during the polymerisation reaction), failing tocomplete a DNA copy of the mRNA template during 1st strand cDNAsynthesis.

[0038] There are several methods available and well known to thoseskilled in the art to obtain full-length cDNAs, or extend short cDNAs,for example those based on the method of Rapid Amplification of cDNAends (RACE) (see, for example, Frohman et al., PNAS USA 85, 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon™ technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs have been prepared from mRNA extracted from a chosentissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the ‘missing’ 5′ endof the cDNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using‘nested’ primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the known gene sequence). The products of thisreaction can then be analysed by DNA sequencing and a full-length cDNAconstructed either by joining the product directly to the existing cDNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

[0039] Recombinant polypeptides of the present invention may be preparedby processes well known in the art from genetically engineered hostcells comprising expression systems. Accordingly, in a further aspect,the present invention relates to expression systems which comprise apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

[0040] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Introduction ofpolynucleotides into host cells can be effected by methods described inmany standard laboratory manuals, such as Davis et al., Basic Methods inMolecular Biology (1986) and Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989). Preferred such methods include, forinstance, calcium phosphate transfection, DEAE-dextran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

[0041] Representative examples of appropriate hosts include bacterialcells, such as Streptococci, Staphylococci, E. coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 andBowes melanoma cells; and plant cells.

[0042] A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0043] If a polypeptide of the present invention is to be expressed foruse in screening assays, it is generally preferred that the polypeptidebe produced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

[0044] Polypeptides of the present invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography is employed forpurification. Well known techniques for refolding proteins may beemployed to regenerate active conformation when the polypeptide isdenatured during intracellular synthesis, isolation and or purification.

[0045] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofthe gene characterised by the polynucleotide of SEQ ID NO:1 which isassociated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredspatial or temporal expression of the gene. Individuals carryingmutations in the gene may be detected at the DNA level by a variety oftechniques.

[0046] Nucleic acids for diagnosis may be obtained from a subject'scells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniquesprior to analysis. RNA or cDNA may also be used in similar fashion.Deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to labeled ANT5nucleotide sequences. Perfectly matched sequences can be distinguishedfrom mismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing (ee, e.g.,Myers et al., Science (1985) 230:1242). Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method (see Cotton etal., Proc Natl Acad Sci USA (1985) 85: 4397-4401). In anotherembodiment, an array of oligonucleotides probes comprising ANT5nucleotide sequence or fragments thereof can be constructed to conductefficient screening of e.g., genetic mutations. Array technology methodsare well known and have general applicability and can be used to addressa variety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability (see for example: M. Chee etal., Science, Vol 274, pp 610-613 (1996)).

[0047] The diagnostic assays offer a process for diagnosing ordetermining a susceptibility to the Diseases through detection ofmutation in the ANT5 gene by the methods described. In addition, suchdiseases may be diagnosed by methods comprising determining from asample derived from a subject an abnormally decreased or increased levelof polypeptide or mRNA. Decreased or increased expression can bemeasured at the RNA level using any of the methods well known in the artfor the quantitation of polynucleotides, such as, for example, nucleicacid amplification, for instance PCR, RT-PCR, RNase protection, Northernblotting and other hybridization methods. Assay techniques that can beused to determine levels of a protein, such as a polypeptide of thepresent invention, in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

[0048] Thus in another aspect, the present invention relates to adiagonostic kit which comprises:

[0049] (a) a polynucleotide of the present invention, preferably thenucleotide sequence of SEQ ID NO:1, or a fragment thereof;

[0050] (b) a nucleotide sequence complementary to that of (a);

[0051] (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO:2 or a fragment thereof; or

[0052] (d) an antibody to a polypeptide of the present invention,preferably to the polypeptide of SEQ ID NO:2.

[0053] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component. Such a kit will be of use indiagnosing a disease or suspectability to a disease, particularlycongestive heart failure, ischaemic heart disease, cardiac arrhytmias,diastolic or systolic dysfunction, hypertrophic cardiomyopathy, stroke,amongst others.

[0054] The nucleotide sequences of the present invention are alsovaluable for chromosome localisation. The sequence is specificallytargeted to, and can hybridize with, a particular location on anindividual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found in, for example, V. McKusick,Mendelian Inheritance in Man (available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

[0055] The differences in the cDNA or genomic sequence between affectedand unaffected individuals can also be determined. If a mutation isobserved in some or all of the affected individuals but not in anynormal individuals, then the mutation is likely to be the causativeagent of the disease.

[0056] The gene of the present invention maps to human chromosome7q21-q22.

[0057] The nucleotide sequences of the present invention are alsovaluable for tissue localisation. Such techniques allow thedetermination of expression patterns of the human ANT5 polypeptides intissues by detection of the mRNAs that encode them. These techniquesinclude in situ hybridziation techniques and nucleotide amplificationtechniques, for example PCR. Such techniques are well known in the art.Results from these studies provide an indication of the normal functionsof the polypeptides in the organism. In addition, comparative studies ofthe normal expression pattern of human ANT5 mRNAs with that of mRNAsencoded by a human ANT5 gene provide valuable insights into the role ofmutant human ANT5 polypeptides, or that of inappropriate expression ofnormal human ANT5 polypeptides, in disease. Such inappropriateexpression may be of a temporal, spatial or simply quantitative nature.

[0058] The polypeptides of the invention or their fragments or analogsthereof, or cells expressing them, can also be used as immunogens toproduce antibodies immunospecific for polypeptides of the presentinvention. The term “immunospecific” means that the antibodies havesubstantially greater affinity for the polypeptides of the inventionthan their affinity for other related polypeptides in the prior art.

[0059] Antibodies generated against polypeptides of the presentinvention may be obtained by administering the polypeptides orepitope-bearing fragments, analogs or cells to an animal, preferably anon-human animal, using routine protocols. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler, G. and Milstein, C., Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridomatechnique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp.77-96, Alan R. Liss, Inc., 1985).

[0060] Techniques for the production of single chain antibodies, such asthose described in U.S. Pat. No. 4,946,778, can also be adapted toproduce single chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

[0061] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptidesby affinity chromatography.

[0062] Antibodies against polypeptides of the present invention may alsobe employed to treat the Diseases, amongst others.

[0063] In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgGl, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

[0064] Another aspect of the invention relates to a method for inducingan immunological response in a mammal which comprises inoculating themammal with a polypeptide of the present invention, adequate to produceantibody and/or T cell immune response to protect said animal from theDiseases hereinbefore mentioned, amongst others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises, delivering a polypeptide of the presentinvention via a vector directing expression of the polynucleotide andcoding for the polypeptide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

[0065] A further aspect of the invention relates to animmunological/vaccine formulation (composition) which, when introducedinto a mammalian host, induces an immunological response in that mammalto a polypeptide of the present invention wherein the compositioncomprises a polypeptide or polynucleotide of the present invention. Thevaccine formulation may further comprise a suitable carrier. Since apolypeptide may be broken down in the stomach, it is preferablyadministered parenterally (for instance, subcutaneous, intramuscular,intravenous, or intradermal injection). Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

[0066] Polypeptides of the present invention are responsible for one ormore biological functions, including one or more disease states, inparticular the Diseases hereinbefore mentioned. It is therefore desirousto devise screening methods to identify compounds which stimulate orwhich inhibit the function of the polypeptide. Accordingly, in a furtheraspect, the present invention provides for a method of screeningcompounds to identify those which stimulate or which inhibit thefunction of the polypeptide. In general, agonists or antagonists may beemployed for therapeutic and prophylactic purposes for such Diseases ashereinbefore mentioned. Compounds may be identified from a variety ofsources, for example, cells, cell-free preparations, chemical libraries,and natural product mixtures. Such agonists, antagonists or inhibitorsso-identified may be natural or modified substrates, ligands, receptors,enzymes, etc., as the case may be, of the polypeptide; or may bestructural or functional mimetics thereof (see Coligan et al., CurrentProtocols in Immunology 1(2):Chapter 5 (1991)).

[0067] The screening method may simply measure the binding of acandidate compound to the polypeptide, or to cells or membranes bearingthe polypeptide, or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound.Alternatively, the screening method may involve competition with alabeled competitor. Further, these screening methods may test whetherthe candidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells bearing the polypeptide. Inhibitors of activation aregenerally assayed in the presence of a known agonist and the effect onactivation by the agonist by the presence of the candidate compound isobserved. Constitutively active polypeptides may be employed inscreening methods for inverse agonists or inhibitors, in the absence ofan agonist or inhibitor, by testing whether the candidate compoundresults in inhibition of activation of the polypeptide. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide of the presentinvention, to form a mixture, measuring ANT5 activity in the mixture,and comparing the ANT5 activity of the mixture to a standard. Fusionproteins, such as those made from Fc portion and ANT5 polypeptide, ashereinbefore described, can also be used for high-throughput screeningassays to identify antagonists for the polypeptide of the presentinvention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); andK. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).

[0068] The polynucleotides, polypeptides and antibodies to thepolypeptide of the present invention may also be used to configurescreening methods for detecting the effect of added compounds on theproduction of mRNA and polypeptide in cells. For example, an ELISA assaymay be constructed for measuring secreted or cell associated levels ofpolypeptide using monoclonal and polyclonal antibodies by standardmethods known in the art. This can be used to discover agents which mayinhibit or enhance the production of polypeptide (also called antagonistor agonist, respectively) from suitably manipulated cells or tissues.

[0069] The polypeptide may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the polypeptide is labeled with aradioactive isotope (for instance, ¹²⁵I), chemically modified (forinstance, biotinylated), or fused to a peptide sequence suitable fordetection or purification, and incubated with a source of the putativereceptor (cells, cell membranes, cell supernatants, tissue extracts,bodily fluids). Other methods include biophysical techniques such assurface plasmon resonance and spectroscopy. These screening methods mayalso be used to identify agonists and antagonists of the polypeptidewhich compete with the binding of the polypeptide to its receptors, ifany. Standard methods for conducting such assays are well understood inthe art.

[0070] Examples of potential polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, enzymes, etc., as thecase may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

[0071] Thus, in another aspect, the present invention relates to ascreening kit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for polypeptides of the present invention; orcompounds which decrease or enhance the production of such polypeptides,which comprises:

[0072] (a) a polypeptide of the present invention;

[0073] (b) a recombinant cell expressing a polypeptide of the presentinvention;

[0074] (c) a cell membrane expressing a polypeptide of the presentinvention; or

[0075] (d) antibody to a polypeptide of the present invention;

[0076] which polypeptide is preferably that of SEQ ID NO:2.

[0077] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component.

[0078] It will be readily appreciated by the skilled artisan that apolypeptide of the present invention may also be used in a method forthe structure-based design of an agonist, antagonist or inhibitor of thepolypeptide, by:

[0079] (a) determining in the first instance the three-dimensionalstructure of the polypeptide;

[0080] (b) deducing the three-dimensional structure for the likelyreactive or binding site(s) of an agonist, antagonist or inhibitor;

[0081] (c) synthesing candidate compounds that are predicted to bind toor react with the deduced binding or reactive site; and

[0082] (d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

[0083] It will be further appreciated that this will normally be aniterative process.

[0084] In a further aspect, the present invention provides methods oftreating abnormal conditions such as, for instance, congestive heartfailure, ischaemic heart disease, cardiac arrhytmias, diastolic orsystolic dysfunction, hypertrophic cardiomyopathy, stroke, related toeither an excess of, or an under-expression of, ANT5 polypeptideactivity.

[0085] If the activity of the polypeptide is in excess, severalapproaches are available. One approach comprises administering to asubject in need thereof an inhibitor compound (antagonist) ashereinabove described, optionally in combination with a pharmaceuticallyacceptable carrier, in an amount effective to inhibit the function ofthe polypeptide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide may be administered. Typical examples of suchcompetitors include fragments of the ANT5 polypeptide.

[0086] In still another approach, expression of the gene encodingendogenous ANT5 polypeptide can be inhibited using expression blockingtechniques. Known such techniques involve the use of antisensesequences, either internally generated or externally administered (see,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides whichform triple helices (“triplexes”) with the gene can be supplied (see,for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney et al.,Science (1988) 241:456; Dervan et al., Science (1991) 251:1360). Theseoligomers can be administered per se or the relevant oligomers can beexpressed in vivo. Synthetic antisense or triplex oligonucleotides maycomprise modified bases or modified backbones. Examples of the latterinclude methylphosphonate, phosphorothioate or peptide nucleic acidbackbones. Such backbones are incorporated in the antisense or triplexoligonucleotide in order to provide protection from degradation bynucleases and are well known in the art. Antisense and triplex moleculessynthesised with these or other modified backbones also form part of thepresent invention.

[0087] In addition, expression of the human ANT5 polypeptide may beprevented by using ribozymes specific to the human ANT5 mRNA sequence.Ribozymes are catalytically active RNAs that can be natural or synthetic(see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4),527-33.) Synthetic ribozymes can be designed to specifically cleavehuman ANT5 mRNAs at selected positions thereby preventing translation ofthe human ANT5 mRNAs into functional polypeptide. Ribozymes may besynthesised with a natural ribose phosphate backbone and natural bases,as normally found in RNA molecules. Alternatively the ribosymes may besynthesised with non-natural backbones to provide protection fromribonuclease degradation, for example, 2′-O-methyl RNA, and may containmodified bases.

[0088] For treating abnormal conditions related to an under-expressionof ANT5 and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates a polypeptide of thepresent invention, i.e., an agonist as described above, in combinationwith a pharmaceutically acceptable carrier, to thereby alleviate theabnormal condition. Alternatively, gene therapy may be employed toeffect the endogenous production of ANT5 by the relevant cells in thesubject. For example, a polynucleotide of the invention may beengineered for expression in a replication defective retroviral vector,as discussed above. The retroviral expression construct may then beisolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding a polypeptide of thepresent invention such that the packaging cell now produces infectiousviral particles containing the gene of interest. These producer cellsmay be administered to a subject for engineering cells in vivo andexpression of the polypeptide in vivo. For an overview of gene therapy,see Chapter 20, Gene Therapy and other Molecular Genetic-basedTherapeutic Approaches, (and references cited therein) in HumanMolecular Genetics, T Strachan and A P Read, BIOS Scientific PublishersLtd (1996). Another approach is to administer a therapeutic amount of apolypeptide of the present invention in combination with a suitablepharmaceutical carrier.

[0089] In a further aspect, the present invention provides forpharmaceutical compositions comprising a therapeutically effectiveamount of a polypeptide, such as the soluble form of a polypeptide ofthe present invention, agonist/antagonist peptide or small moleculecompound, in combination with a pharmaceutically acceptable carrier orexcipient. Such carriers include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0090] The composition will be adapted to the route of administration,for instance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

[0091] The dosage range required depends on the choice of peptide orother compounds of the present invention, the route of administration,the nature of the formulation, the nature of the subject's condition,and the judgment of the attending practitioner. Suitable dosages,however, are in the range of 0.1-100 μg/kg of subject. Wide variationsin the needed dosage, however, are to be expected in view of the varietyof compounds available and the differing efficiencies of various routesof administration. For example, oral administration would be expected torequire higher dosages than administration by intravenous injection.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

[0092] Polypeptides used in treatment can also be generated endogenouslyin the subject, in treatment modalities often referred to as “genetherapy” as described above. Thus, for example, cells from a subject maybe engineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

[0093] Polynucleotide and polypeptide sequences form a valuableinformation resource with which to identify further sequences of similarhomology. This is most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data to search asequence database using well known searching tools, such as those in theGCG and Lasergene software packages. Accordingly, in a further aspect,the present invention provides for a computer readable medium havingstored thereon a polynucleotide comprising the sequence of SEQ ID NO:1and/or a polypeptide sequence encoded thereby.

[0094] The following definitions are provided to facilitateunderstanding of certain terms used frequently hereinbefore.

[0095] “Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

[0096] “Isolated” means altered “by the hand of man” from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated”, as the term is employed herein.

[0097] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0098] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as post-translational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance,PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993; Wold, F., Post-translationalProtein Modifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York, 1983; Seifter et al., “Analysis for proteinmodifications and nonprotein cofactors”, Meth Enzymol (1990) 182:626-646and Rattan et al., “Protein Synthesis: Post-translational Modificationsand Aging”, Ann NY Acad Sci (1992) 663:48-62).

[0099] “Variant” refers to a polynucleotide or polypeptide that differsfrom a reference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

[0100] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(l):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

[0101] Preferred parameters for polypeptide sequence comparison includethe following:

[0102] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0103] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci.

[0104] USA. 89:10915-10919 (1992)

[0105] Gap Penalty: 12

[0106] Gap Length Penalty: 4

[0107] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0108] Preferred parameters for polynucleotide comparison include thefollowing:

[0109] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0110] Comparison matrix: matches=+10, mismatch=0

[0111] Gap Penalty: 50

[0112] Gap Length Penalty: 3

[0113] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0114] By way of example, a polynucleotide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:1,that is be 100% identical, or it may include up to a certain integernumber of nucleotide alterations as compared to the reference sequence.Such alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in SEQ ID NO:1 by the numerical percent of the respectivepercent identity(divided by 100) and subtracting that product from saidtotal number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

[0115] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, and y is, for instance,0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%. 0.95 for95%,etc., and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

[0116] Similarly, a polypeptide sequence of the present invention may beidentical to the reference sequence of SEQ ID NO:2, that is be 100%identical, or it may include up to a certain integer number of aminoacid alterations as compared to the reference sequence such that the %identity is less than 100%. Such alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of amino acidalterations for a given % identity is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the numerical percent ofthe respective percent identity(divided by 100) and then subtractingthat product from said total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

[0117] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, and y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein anynon-integer product of x_(a) and y is rounded down to the nearestinteger prior to subtracting it from x_(a).

[0118] “Homolog” is a generic term used in the art to indicate apolynucleotide or polypeptide sequence possessing a high degree ofsequence relatedness to a subject sequence. Such relatedness may bequantified by determining the degree of identity and/or similaritybetween the sequences being compared as hereinbefore described. Fallingwithin this generic term are the terms “ortholog”, meaning apolynucleotide or polypeptide that is the functional equivalent of apolynucleotide or polypeptide in another species, and “paralog” meaninga functionally similar sequence when considered within the same species.

[0119] “Fusion protein” refers to a protein encoded by two, oftenunrelated, fused genes or fragments thereof. In one example, EP-A-0 464discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232 262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

[0120] All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth. SEQUENCE INFORMATION SEQ ID NO:1AGCCGCCCGGGTCCCAAACGCCAGCCAGCCAGTCAGTGGGTCCCGCAGTCGCCCGCAACCGGGGCGAATCATGGCGGCCGCAAGGGTGGCTTTAACCAAGAGAGCAGATCCAGCTGAGCTTAGAACAATATTTTTGAAGTATGCAAGCATTGAGAAAAACGGTGAATTTTTCATGTCCCCCAATGACTTTGTCACTCGATACTTGAACATTTTTGGAGAAAGCCAGCCTAATCCAAAGACTGTGGAACTTTTAAGTGGAgTGGTGGATCAGACCAAAGATGGATTAATATCTTTTCAtGAATTTGTTGCCTTTGAATCTGTCCTGTGTGCCCCTGATGCTTTGTTTATGGTAGCCTTTCAGCTGTTTGACAAAGCTGGCAAAGGAGAAGTAACTTTTGAGGATGTTAAGCAAGTTTTTGGACAGACCACAATTCATCAACATATTCCATTTAACTGGGATTCAGAATTTGTGCAACTACATTTTGGAAAAGAAAGAAAAaGACACCTGACATATGCGGAATTTACTCAGTTTTTATTGGAAATACAACTGGAGCACGCAAAGCAAGCCTTTGTGCAACGGGACAATGCTAGGACTGGGAGAGTCACAGCCATCGACTTCCGAGACATCATGGTCACCATCCGCCCCCATGTCTTGACTCCTTTTGTAGAAGAATGTCTAGTAGCTGCTGCTGGAGGTACCACATCCCATCAAGTTAGTTTCTCCTATTTTAATGGATTTAATTCGCTCCTTAACAACATGGAACTCATTAGAAAGATCTATAGCACTCTGGCTGGCACCAGGAAAGATGTTGAAGTGACTAAGGAGGAGTTTGTTCTGGCAGCTCAGAAATTTGGTCAGGTTACACCCATGGAAGTTGACATCTTGTTTCAGTTAGCAGATTTATATGAGCCAAGGGGACGTATGACCTTAGCAGACATTGAACGGATTGCTCCTCTGGAAGAGGGAACTCTGCCCTTTAACTTGGCTGAGGCCCAGAGGCAGGCCTCAGGTGATTCAGCTCGACCAGTTCTTCTACAAGTTGCAGAGTCGGCCTACAGGTTTGGTCTGGGTTCTGTTGCTGGAGCTGTTGGAGCCACTGCTGTGTATCCTATCGATCTTGTAAAAACTCGAATGCAGAACCAACGATCAACTGGCTCTTTTGTGGGAGAACTCATGTATAAAAACAGCTTTGACTGTTTTAAGAAAGTGCTACGCTATGAAGGCTTCTTTGGACTGTATAGAGGTCTGTTGCCACAGTTATTGGGAGTTGCCCCAGAGAAGGCCATAAAACTTACAGTGAACGATTTTGTGAGGGATAAATTTATGCACAAAGATGGTTCGGTCCCACTTGCAGCAGAAATTCTTGCTGGAGGCTGCGCTGGAGGCTCCCAGGTGATTTTCACAAATCCTTTAGAAATCGTCAAGATCCGTTTGCAAGTGGCAGGAGAAATCACCACTGGTCCTCGAGTCAGTGCTCTGTCTGTCGTGCGGGACCTGGGGTTTTTTGGGATCTACAAGGGTGCCAAAGCATGCTTTCTGCGGGACATTCCTTTCTCGGCCATCTACTTTCCGTGCTATGCTCATGTGAAGGCTTCCTTTGCAAATGAAGATGGGCAGGTTAGCCCAGGAAGCCTGCTCTTAGCTGGTGCCATAGCTGGTATGCCTGCAGCATCTTTAGTGACCCCTGCTGATGTTATCAAGACGAGATTACAGGTGGCTGCCCGGGCTGGCCAAACCACTTACAGCGGAGTGATAGACTGCTTTAGAAAGATACTGCGTGAAGAAGGACCAAAAGCTCTGTGGAAGGGAGCTGGTGCTCGTGTATTTCGATCCTCACCCCAGTTTGGTGTAACTTTGCTGACTTACGAATTGCTACAGCGATGGTTCTACATTGATTTTGGAGGAGTAAAACCCATGGGATCAGAGCCAGTTCCTAAATCCAGGATCAACCTGCCTGCCCCGAATCCTGATCACGTTGGGGGCTACAAACTGGCAGTTGCTACATTTGCAGGGATTGAAAACAAATTTGGACTTTACCTACCTCTCTTCAAGCCATCAGTATCTACCTCAAAGGCTATTGGTGGAGGCCCATAG SEQ ID NO:2MAAARVALTKRADPAELRTIFLKYASIEKNGEFFMSPNDFVTRYLNIFGESQPNPKTVELLSGVVDQTKDGLISFHEFVAFESVLCAPDALFMVAFQLFDKAGKGEVTFEDVKQVFGQTTIHQHIPFNWDSEFVQLHFGKERKRHLTYAEFTQFLLEIQLEHAKQAFVQRDNARTGRVTAIDFRDIMVTIRPHVLTPFVEECLVAAAGGTTSHQVSFSYFNGFNSLLNNMELIRKIYSTLAGTRKDVEVTKEEFVLAAQKFGQVTPMEVDILFQLADLYEPRGRMTLADIERIAPLEEGTLPFNLAEAQRQASGDSARPVLLQVAESAYRFGLGSVAGAVGATAVYPIDLVKTRMQNQRSTGSFVGELMYKNSFDCFKKVLRYEGFFGLYRGLLPQLLGVAPEKAIKLTVNDFVRDKFMHKDGSVPLAAEILAGGCAGGSQVIFTNPLEIVKIRLQVAGEITTGPRVSALSVVRDLGFFGIYKGAKACFLRDIPFSAIYFPCYAHVKASFANEDGQVSPGSLLLAGAIAGMPAASLVTPADVIKTRLQVAARAGQTTYSGVIDCFRKILREEGPKALWKGAGARVFRSSPQFGVTLLTYELLQRWFYIDFGGVKPMGSEPVPKSRINLPAPNPDHVGGYKLAVATFAGIENKFGLYLPLFKPSVSTSKAIGGGP SEQ ID NO:3TCGACCCACGCGTCCGATTTATTTGAGGCTGCTGGAGGTACCACATCCCATCAAGTTAGTTTCTCCTATTTTAATGGATTTAATTCGCTCCTTAACAACATGGAACTCATTAGAAAGATCTATAGCACGCTGGCTGGCACCAGGAAAGATGTTGAAGTGACTAAGGAGGAGTTTGTTCTGGCAGCTCAGAAATTTGGTCAGGTTACACCCATGGAAGTTGACATCTTGTTTCAGTTAGCAGATTTATATGAGCCAAGGGGACGTATGACCTTAGCAGACATTGAACGGATTGCTCCTCTGGAAGAGGGAACTCTGCCCTTTAACTTGGCTGAGGCCCAGAGGCAGCAGAAGGCCTCAGGTGATTCAGCTCGACCAGTTCTTCTACAAGTTGCAGAGTCGGCCTACAGGTTTGGTCTGGGTTCTGTTGCTGGAGCTGTTGGAGCCACTGCTGTGTATCCTATCGATCTTGTAAAAACTCGAATGCAGAACCAACGATCAACTGGCTCTTTTGTGGGAGAACTCATGTATAAAAACAGCTTTGACTGTTTTAAGAAAGTGCTACGCTATGAAGGCTTCTTTGGACTGTATAGAGGTCTGTTGCCACAGTTATTGGGAGTTGCCCCAGAGAAGGCCATAAAACTTACAGTGAACGATTTTGTGAGGGATAAATTTATGCACAAAGATGGTTCGGTCCCACTTGCAGCAGAAATTCTTGCTGGAGGCTGCGCTGGAGGCTCCCAGGTGATTTTCACAAATCCTTTAGAAATCGTCAAGATCCGTTTGCAAGTGGCAGGAGAAATCACCACTGGTCCTCGAGTCAGTGCTCTGTCTGTCGTGCGGGACCTGGGGTTTTTTGGGATCTACAAGGGTGCCAAAGCATGCTTTCTGCGGGACATTCCTTTCTCGGCCATCTACTTTCCGTGCTATGCTCATGTGAAGGCTTCCTTTGCAAATGAAGATGGGCAGGTTAGCCCAGGAAGCCTGCTCTTAGCTGGTGCCATAGCTGGTATGCCTGCAGCATCTTTAGTGACCCCTGCTGATGTTATCAAGACGAGATTACAGGTGGCTGCCCGGGCTGGCCAAACCACTTACAGCGGAGTGATAGACTGCTTTAGAAAGATACTGCGTGAAGAAGGACCAAAAGCTCTGTGGAAGGGAGCTGGTGCTCGTGTATTTCGATCCTCACCCCAGTTTGGTGTAACTTTGCTGACTTACGAATTGCTACAGCGATGGTTCTACATTGATTTTGGAGGAGTAAAACCCATGGGATCAGAGCCAGTTCCTAAATCCAGGATCAACCTGCCTGCCCCGAATCCTGATCACGTTGGGGGCTACAAACTGGCAGTTGCTACATTTGCAGGGATTGAAAACAAATTTGGACTTTACCTACCTCTCTTCAAGCCATCAGTATCTACCTCAAAGGCTATTGGTGGAGGCCCATAG SEQ ID NO:4MELIRKIYSTLAGTRKDVEVTKEEFVLAAQKFGQVTPMEVDILFQLADLYEPRGRMTLADIERIAPLEEGTLPFNLAEAQRQQKASGDSARPVLLQVAESAYRFGLGSVAGAVGATAVYPIDLVKTRMQNQRSTGSFVGELMYKNSFDCFKKVLRYEGFFGLYRGLLPQLLGVAPEKAIKLTVNDFVRDKFMHKDGSVPLAAEILAGGCAGGSQVIFTNPLEIVKIRLQVAGEITTGPRVSALSVVRDLGFFGIYKGAKACFLRDIPFSAIYFPCYAHVKASFANEDGQVSPGSLLLAGAIAGMPAASLVTPADVIKTRLQVAARAGQTTYSGVIDCFRKILREEGPKALWKGAGARVFRSSPQFGVTLLTYELLQRWFYIDFGGVKPMGSEPVPKSRINLPAPNPDHVGGYKLAVATFAGIENKFGLYLPLFKPSVSTSKAIGGGP

[0121]

1 4 1 2095 DNA HOMO SAPIENS 1 agccgcccgg gtcccaaacg ccagccagccagtcagtggg tcccgcagtc gcccgcaacc 60 ggggcgaatc atggcggccg caagggtggctttaaccaag agagcagatc cagctgagct 120 tagaacaata tttttgaagt atgcaagcattgagaaaaac ggtgaatttt tcatgtcccc 180 caatgacttt gtcactcgat acttgaacatttttggagaa agccagccta atccaaagac 240 tgtggaactt ttaagtggag tggtggatcagaccaaagat ggattaatat cttttcatga 300 atttgttgcc tttgaatctg tcctgtgtgcccctgatgct ttgtttatgg tagcctttca 360 gctgtttgac aaagctggca aaggagaagtaacttttgag gatgttaagc aagtttttgg 420 acagaccaca attcatcaac atattccatttaactgggat tcagaatttg tgcaactaca 480 ttttggaaaa gaaagaaaaa gacacctgacatatgcggaa tttactcagt ttttattgga 540 aatacaactg gagcacgcaa agcaagcctttgtgcaacgg gacaatgcta ggactgggag 600 agtcacagcc atcgacttcc gagacatcatggtcaccatc cgcccccatg tcttgactcc 660 ttttgtagaa gaatgtctag tagctgctgctggaggtacc acatcccatc aagttagttt 720 ctcctatttt aatggattta attcgctccttaacaacatg gaactcatta gaaagatcta 780 tagcactctg gctggcacca ggaaagatgttgaagtgact aaggaggagt ttgttctggc 840 agctcagaaa tttggtcagg ttacacccatggaagttgac atcttgtttc agttagcaga 900 tttatatgag ccaaggggac gtatgaccttagcagacatt gaacggattg ctcctctgga 960 agagggaact ctgcccttta acttggctgaggcccagagg caggcctcag gtgattcagc 1020 tcgaccagtt cttctacaag ttgcagagtcggcctacagg tttggtctgg gttctgttgc 1080 tggagctgtt ggagccactg ctgtgtatcctatcgatctt gtaaaaactc gaatgcagaa 1140 ccaacgatca actggctctt ttgtgggagaactcatgtat aaaaacagct ttgactgttt 1200 taagaaagtg ctacgctatg aaggcttctttggactgtat agaggtctgt tgccacagtt 1260 attgggagtt gccccagaga aggccataaaacttacagtg aacgattttg tgagggataa 1320 atttatgcac aaagatggtt cggtcccacttgcagcagaa attcttgctg gaggctgcgc 1380 tggaggctcc caggtgattt tcacaaatcctttagaaatc gtcaagatcc gtttgcaagt 1440 ggcaggagaa atcaccactg gtcctcgagtcagtgctctg tctgtcgtgc gggacctggg 1500 gttttttggg atctacaagg gtgccaaagcatgctttctg cgggacattc ctttctcggc 1560 catctacttt ccgtgctatg ctcatgtgaaggcttccttt gcaaatgaag atgggcaggt 1620 tagcccagga agcctgctct tagctggtgccatagctggt atgcctgcag catctttagt 1680 gacccctgct gatgttatca agacgagattacaggtggct gcccgggctg gccaaaccac 1740 ttacagcgga gtgatagact gctttagaaagatactgcgt gaagaaggac caaaagctct 1800 gtggaaggga gctggtgctc gtgtatttcgatcctcaccc cagtttggtg taactttgct 1860 gacttacgaa ttgctacagc gatggttctacattgatttt ggaggagtaa aacccatggg 1920 atcagagcca gttcctaaat ccaggatcaacctgcctgcc ccgaatcctg atcacgttgg 1980 gggctacaaa ctggcagttg ctacatttgcagggattgaa aacaaatttg gactttacct 2040 acctctcttc aagccatcag tatctacctcaaaggctatt ggtggaggcc catag 2095 2 674 PRT HOMO SAPIENS 2 Met Ala AlaAla Arg Val Ala Leu Thr Lys Arg Ala Asp Pro Ala Glu 1 5 10 15 Leu ArgThr Ile Phe Leu Lys Tyr Ala Ser Ile Glu Lys Asn Gly Glu 20 25 30 Phe PheMet Ser Pro Asn Asp Phe Val Thr Arg Tyr Leu Asn Ile Phe 35 40 45 Gly GluSer Gln Pro Asn Pro Lys Thr Val Glu Leu Leu Ser Gly Val 50 55 60 Val AspGln Thr Lys Asp Gly Leu Ile Ser Phe His Glu Phe Val Ala 65 70 75 80 PheGlu Ser Val Leu Cys Ala Pro Asp Ala Leu Phe Met Val Ala Phe 85 90 95 GlnLeu Phe Asp Lys Ala Gly Lys Gly Glu Val Thr Phe Glu Asp Val 100 105 110Lys Gln Val Phe Gly Gln Thr Thr Ile His Gln His Ile Pro Phe Asn 115 120125 Trp Asp Ser Glu Phe Val Gln Leu His Phe Gly Lys Glu Arg Lys Arg 130135 140 His Leu Thr Tyr Ala Glu Phe Thr Gln Phe Leu Leu Glu Ile Gln Leu145 150 155 160 Glu His Ala Lys Gln Ala Phe Val Gln Arg Asp Asn Ala ArgThr Gly 165 170 175 Arg Val Thr Ala Ile Asp Phe Arg Asp Ile Met Val ThrIle Arg Pro 180 185 190 His Val Leu Thr Pro Phe Val Glu Glu Cys Leu ValAla Ala Ala Gly 195 200 205 Gly Thr Thr Ser His Gln Val Ser Phe Ser TyrPhe Asn Gly Phe Asn 210 215 220 Ser Leu Leu Asn Asn Met Glu Leu Ile ArgLys Ile Tyr Ser Thr Leu 225 230 235 240 Ala Gly Thr Arg Lys Asp Val GluVal Thr Lys Glu Glu Phe Val Leu 245 250 255 Ala Ala Gln Lys Phe Gly GlnVal Thr Pro Met Glu Val Asp Ile Leu 260 265 270 Phe Gln Leu Ala Asp LeuTyr Glu Pro Arg Gly Arg Met Thr Leu Ala 275 280 285 Asp Ile Glu Arg IleAla Pro Leu Glu Glu Gly Thr Leu Pro Phe Asn 290 295 300 Leu Ala Glu AlaGln Arg Gln Ala Ser Gly Asp Ser Ala Arg Pro Val 305 310 315 320 Leu LeuGln Val Ala Glu Ser Ala Tyr Arg Phe Gly Leu Gly Ser Val 325 330 335 AlaGly Ala Val Gly Ala Thr Ala Val Tyr Pro Ile Asp Leu Val Lys 340 345 350Thr Arg Met Gln Asn Gln Arg Ser Thr Gly Ser Phe Val Gly Glu Leu 355 360365 Met Tyr Lys Asn Ser Phe Asp Cys Phe Lys Lys Val Leu Arg Tyr Glu 370375 380 Gly Phe Phe Gly Leu Tyr Arg Gly Leu Leu Pro Gln Leu Leu Gly Val385 390 395 400 Ala Pro Glu Lys Ala Ile Lys Leu Thr Val Asn Asp Phe ValArg Asp 405 410 415 Lys Phe Met His Lys Asp Gly Ser Val Pro Leu Ala AlaGlu Ile Leu 420 425 430 Ala Gly Gly Cys Ala Gly Gly Ser Gln Val Ile PheThr Asn Pro Leu 435 440 445 Glu Ile Val Lys Ile Arg Leu Gln Val Ala GlyGlu Ile Thr Thr Gly 450 455 460 Pro Arg Val Ser Ala Leu Ser Val Val ArgAsp Leu Gly Phe Phe Gly 465 470 475 480 Ile Tyr Lys Gly Ala Lys Ala CysPhe Leu Arg Asp Ile Pro Phe Ser 485 490 495 Ala Ile Tyr Phe Pro Cys TyrAla His Val Lys Ala Ser Phe Ala Asn 500 505 510 Glu Asp Gly Gln Val SerPro Gly Ser Leu Leu Leu Ala Gly Ala Ile 515 520 525 Ala Gly Met Pro AlaAla Ser Leu Val Thr Pro Ala Asp Val Ile Lys 530 535 540 Thr Arg Leu GlnVal Ala Ala Arg Ala Gly Gln Thr Thr Tyr Ser Gly 545 550 555 560 Val IleAsp Cys Phe Arg Lys Ile Leu Arg Glu Glu Gly Pro Lys Ala 565 570 575 LeuTrp Lys Gly Ala Gly Ala Arg Val Phe Arg Ser Ser Pro Gln Phe 580 585 590Gly Val Thr Leu Leu Thr Tyr Glu Leu Leu Gln Arg Trp Phe Tyr Ile 595 600605 Asp Phe Gly Gly Val Lys Pro Met Gly Ser Glu Pro Val Pro Lys Ser 610615 620 Arg Ile Asn Leu Pro Ala Pro Asn Pro Asp His Val Gly Gly Tyr Lys625 630 635 640 Leu Ala Val Ala Thr Phe Ala Gly Ile Glu Asn Lys Phe GlyLeu Tyr 645 650 655 Leu Pro Leu Phe Lys Pro Ser Val Ser Thr Ser Lys AlaIle Gly Gly 660 665 670 Gly Pro 3 1443 DNA HOMO SAPIENS 3 tcgacccacgcgtccgattt atttgaggct gctggaggta ccacatccca tcaagttagt 60 ttctcctattttaatggatt taattcgctc cttaacaaca tggaactcat tagaaagatc 120 tatagcacgctggctggcac caggaaagat gttgaagtga ctaaggagga gtttgttctg 180 gcagctcagaaatttggtca ggttacaccc atggaagttg acatcttgtt tcagttagca 240 gatttatatgagccaagggg acgtatgacc ttagcagaca ttgaacggat tgctcctctg 300 gaagagggaactctgccctt taacttggct gaggcccaga ggcagcagaa ggcctcaggt 360 gattcagctcgaccagttct tctacaagtt gcagagtcgg cctacaggtt tggtctgggt 420 tctgttgctggagctgttgg agccactgct gtgtatccta tcgatcttgt aaaaactcga 480 atgcagaaccaacgatcaac tggctctttt gtgggagaac tcatgtataa aaacagcttt 540 gactgttttaagaaagtgct acgctatgaa ggcttctttg gactgtatag aggtctgttg 600 ccacagttattgggagttgc cccagagaag gccataaaac ttacagtgaa cgattttgtg 660 agggataaatttatgcacaa agatggttcg gtcccacttg cagcagaaat tcttgctgga 720 ggctgcgctggaggctccca ggtgattttc acaaatcctt tagaaatcgt caagatccgt 780 ttgcaagtggcaggagaaat caccactggt cctcgagtca gtgctctgtc tgtcgtgcgg 840 gacctggggttttttgggat ctacaagggt gccaaagcat gctttctgcg ggacattcct 900 ttctcggccatctactttcc gtgctatgct catgtgaagg cttcctttgc aaatgaagat 960 gggcaggttagcccaggaag cctgctctta gctggtgcca tagctggtat gcctgcagca 1020 tctttagtgacccctgctga tgttatcaag acgagattac aggtggctgc ccgggctggc 1080 caaaccacttacagcggagt gatagactgc tttagaaaga tactgcgtga agaaggacca 1140 aaagctctgtggaagggagc tggtgctcgt gtatttcgat cctcacccca gtttggtgta 1200 actttgctgacttacgaatt gctacagcga tggttctaca ttgattttgg aggagtaaaa 1260 cccatgggatcagagccagt tcctaaatcc aggatcaacc tgcctgcccc gaatcctgat 1320 cacgttgggggctacaaact ggcagttgct acatttgcag ggattgaaaa caaatttgga 1380 ctttacctacctctcttcaa gccatcagta tctacctcaa aggctattgg tggaggccca 1440 tag 1443 4447 PRT HOMO SAPIENS 4 Met Glu Leu Ile Arg Lys Ile Tyr Ser Thr Leu AlaGly Thr Arg Lys 1 5 10 15 Asp Val Glu Val Thr Lys Glu Glu Phe Val LeuAla Ala Gln Lys Phe 20 25 30 Gly Gln Val Thr Pro Met Glu Val Asp Ile LeuPhe Gln Leu Ala Asp 35 40 45 Leu Tyr Glu Pro Arg Gly Arg Met Thr Leu AlaAsp Ile Glu Arg Ile 50 55 60 Ala Pro Leu Glu Glu Gly Thr Leu Pro Phe AsnLeu Ala Glu Ala Gln 65 70 75 80 Arg Gln Gln Lys Ala Ser Gly Asp Ser AlaArg Pro Val Leu Leu Gln 85 90 95 Val Ala Glu Ser Ala Tyr Arg Phe Gly LeuGly Ser Val Ala Gly Ala 100 105 110 Val Gly Ala Thr Ala Val Tyr Pro IleAsp Leu Val Lys Thr Arg Met 115 120 125 Gln Asn Gln Arg Ser Thr Gly SerPhe Val Gly Glu Leu Met Tyr Lys 130 135 140 Asn Ser Phe Asp Cys Phe LysLys Val Leu Arg Tyr Glu Gly Phe Phe 145 150 155 160 Gly Leu Tyr Arg GlyLeu Leu Pro Gln Leu Leu Gly Val Ala Pro Glu 165 170 175 Lys Ala Ile LysLeu Thr Val Asn Asp Phe Val Arg Asp Lys Phe Met 180 185 190 His Lys AspGly Ser Val Pro Leu Ala Ala Glu Ile Leu Ala Gly Gly 195 200 205 Cys AlaGly Gly Ser Gln Val Ile Phe Thr Asn Pro Leu Glu Ile Val 210 215 220 LysIle Arg Leu Gln Val Ala Gly Glu Ile Thr Thr Gly Pro Arg Val 225 230 235240 Ser Ala Leu Ser Val Val Arg Asp Leu Gly Phe Phe Gly Ile Tyr Lys 245250 255 Gly Ala Lys Ala Cys Phe Leu Arg Asp Ile Pro Phe Ser Ala Ile Tyr260 265 270 Phe Pro Cys Tyr Ala His Val Lys Ala Ser Phe Ala Asn Glu AspGly 275 280 285 Gln Val Ser Pro Gly Ser Leu Leu Leu Ala Gly Ala Ile AlaGly Met 290 295 300 Pro Ala Ala Ser Leu Val Thr Pro Ala Asp Val Ile LysThr Arg Leu 305 310 315 320 Gln Val Ala Ala Arg Ala Gly Gln Thr Thr TyrSer Gly Val Ile Asp 325 330 335 Cys Phe Arg Lys Ile Leu Arg Glu Glu GlyPro Lys Ala Leu Trp Lys 340 345 350 Gly Ala Gly Ala Arg Val Phe Arg SerSer Pro Gln Phe Gly Val Thr 355 360 365 Leu Leu Thr Tyr Glu Leu Leu GlnArg Trp Phe Tyr Ile Asp Phe Gly 370 375 380 Gly Val Lys Pro Met Gly SerGlu Pro Val Pro Lys Ser Arg Ile Asn 385 390 395 400 Leu Pro Ala Pro AsnPro Asp His Val Gly Gly Tyr Lys Leu Ala Val 405 410 415 Ala Thr Phe AlaGly Ile Glu Asn Lys Phe Gly Leu Tyr Leu Pro Leu 420 425 430 Phe Lys ProSer Val Ser Thr Ser Lys Ala Ile Gly Gly Gly Pro 435 440 445

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidsequence selected from the group having at least: (a) 70% identity; (b)80% identity; (c) 95% identity; or (d) 95% identity to the amino acidsequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (ii) anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:2or (iii) an isolated polypeptide which is the amino acid sequence of SEQID NO:2.
 2. An isolated polynucleotide selected from the groupconsisting of: (i) an isolated polynucleotide comprising a nucleotidesequence encoding a polypeptide that has at least (a) 70% identity; (b)80% identity; (c) 90% identity; or (d) 95% identity; to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2; (ii) anisolated polynucleotide comprising a nucleotide sequence that has atleast: (a) 70% identity (b) 80% identity; (c) 90% identity; or (d) 95%identity; over its entire length to a nucleotide sequence encoding thepolypeptide of SEQ ID NO:2; (iii) an isolated polynucleotide comprisinga nucleotide sequence which has at least; (a) 70% identity; (b) 80%identity; (c) 90% identity; or (d) 95% identity; to that of SEQ ID NO:1over the entire length of SEQ ID NO:1; (iv) an isolated polynucleotidecomprising a nucleotide sequence encoding the polypeptide of SEQ IDNO:2; (vi) an isolated polynucleotide which is the polynucleotide of SEQID NO:1; or (vi) an isolated polynucleotide obtainable by screening anappropriate library under stringent hybridization conditions with alabeled probe having the sequence of SEQ ID NO:1 or a fragment thereof.;or a nucleotide sequence complementary to said isolated polynucleotide.3. An antibody immunospecific for the polypeptide of claim
 1. 4. Amethod for the treatment of a subject: (i) in need of enhanced activityor expression of the polypeptide of claim 1 comprising: (a)administering to the subject a therapeutically effective amount of anagonist to said polypeptide; and/or (b) providing to the subject anisolated polynucleotide comprising a nucleotide sequence encoding saidpolypetide in a form so as to effect production of said polypeptideactivity In vivo.; or (ii) having need to inhibit activity or expressionof the polypeptide of claim 1 comprising: (a) administering to thesubject a therapeutically effective amount of an antagonist to saidpolypeptide; and/or (b) administering to the subject a nucleic acidmolecule that inhibits the expression of a nucleotide sequence encodingsaid polypeptide; and/or (c) administering to the subject atherapeutically effective amount of a polypeptide that competes withsaid polypeptide for its ligand, substrate , or receptor.
 5. A processfor diagnosing a disease or a susceptibility to a disease in a subjectrelated to expression or activity of the polypeptide of claim 1 in asubject comprising: (a) determining the presence or absence of amutation in the nucleotide sequence encoding said polypeptide in thegenome of said subject; and/or (b) analyzing for the presence or amountof said polypeptide expression in a sample derived from said subject. 6.A method for screening to identify compounds which stimulate or whichinhibit the function of the polypeptide of claim 1 which comprises amethod selected from the group consisting of: (a) measuring the bindingof a candidate compound to the polypeptide (or to the cells or membranesbearing the polypeptide) or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound; (b)measuring the binding of a candidate compound to the polypeptide (or tothe cells or membranes bearing the polypeptide) or a fusion proteinthereof in the presense of a labeled competitor; (c) testing whether thecandidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells or cell membranes bearing the polypeptide; (d) mixing acandidate compound with a solution containing a polypeptide of claim 1,to form a mixture, measuring activity of the polypeptide in the mixture,and comparing the activity of the mixture to a standard; or (e)detecting the effect of a candidate compound on the production of mRNAencoding said polypeptide and said polypeptide in cells, using forinstance, an ELISA assay.
 7. An agonist or an antagonist of thepolypeptide of claim
 1. 8. An expression system comprising apolynucleotide capable of producing a polypeptide of claim 1 when saidexpression system is present in a compatible host cell.
 9. A process forproducing a recombinant host cell comprising transforming ortransfecting a cell with the expression system of claim 8 such the thehost cell, under appropriate culture conditions, produces a polypeptidecomprising an amino acid sequence having at least 70% identity to theamino acid sequence of SEQ ID NO:2 over the entire length of SEQ IDNO:2.
 10. A recombinant host cell produced by the process of claim 9.11. A membrane of a recombinant host cell of claim 10 expressing apolypeptide comprising an amino acid sequence having at least 70%identity to the amino acid sequence of SEQ ID NO:2 over the entirelength of SEQ ID NO:2.
 12. A process for producing a polypeptidecomprising culturing a host cell of claim 10 under conditions sufficientfor the production of said polypeptide and recovering the polypeptidefrom the culture.
 13. An isolated polynucleotide selected form the groupconsisting of: (a) an isolated polynucleotide comprising a nucleotidesequence which has at least 70%, 80%, 90%, 95%, 97% identity to SEQ IDNO:3 over the entire length of SEQ ID NO:3; (b) an isolatedpolynucleotide comprising the polynucleotide of SEQ ID NO:3; (c) thepolynucleotide of SEQ ID NO:3; or (d) an isolated polynucleotidecomprising a nucleotide sequence encoding a polypeptide which has atleast 70%,80%, 90%, 95%, 97-99% identity to the amino acid sequence ofSEQ ID NO:4, over the entire length of SEQ ID NO:4.
 14. A polypeptideselected from the group consisting of: (a) a polypeptide which comprisesan amino acid sequence which has at least 70%,80%,90%, 95%, 97-99%identity to that of SEQ ID NO:4 over the entire length of SEQ ID NO:4;(b) a polypeptide which has an amino acid sequence which is at least70%, 80%, 90%, 95%, 97-99% identity to the amino acid sequence of SEQ IDNO:4 over the entire length of SEQ ID NO:4; (c) a polypeptide whichcomprises the amino acid of SEQ ID NO:4; (d) a polypeptide which is thepolypeptide of SEQ ID NO:4; or (e) a polypeptide which is encoded by apolynucleotide comprising the sequence contained in SEQ ID NO:3.